PROJECT SUMMARY/ABSTRACT The goal of this proposal is to address fundamental gaps in our understanding of HER2 receptor tyrosine kinase (RTK) activation mechanism and regulation through direct structural and biophysical studies on nearly full-length receptor. Aberrant HER2 signaling through amplification or oncogenic mutations is at the root of many cancers and remains a key target of therapies. HER2, together with EGFR, HER3, and HER4 comprise the Human Epidermal Growth Factor Receptor (HER) family of RTKs, indispensable for cellular homeostasis. These receptors convert extracellular cues into intracellular responses through homo- and hetero- oligomerization induced by growth factor binding. HER2 distinguishes itself as an orphan receptor with no known ligand and signals by heterodimerization with other members of the HER family. We do not understand how HER2 regulates its catalytic activity in the absence of ligand-bound co-receptors, but many HER2 oncogenic mutations compromise these mechanisms and confer activity in a co-receptor independent manner. Several of those mutations fall outside of the kinase domain, but in the absence of structural understanding of how growth factor binding on the extracellular side of the receptor increases the catalytic activity of the receptor?s intracellular kinase domain, we cannot predict how these mutations elevate HER2 signaling, and most importantly change HER2 vulnerability to known therapeutics. We hypothesize that the orphan receptor HER2 features intrinsic structural mechanisms to regulate catalytic activity in the absence of ligand or co- receptor and oncogenic mutations outside of the kinase domain overcome these regulatory mechanisms via alterations in oligomerization or conformational states to produce aberrant activation. Addressing our hypotheses relies on biophysical analyses of the receptor as a whole. In Aim 1 we seek to determine a high-resolution structure of near-full length HER2 by cryo-electron microscopy (cryo-EM). The lack of any high-resolution structure of a full-length RTK is attributed to challenges in expressing, purifying, and stabilizing a homogeneous receptor sample. We have recently overcome these challenges for HER2 by engineering a near-full length HER2 construct that is robustly expressed and purified in a stable form. Our preliminary negative stain-electron microscopy imaging demonstrates high sample homogeneity that permits structural investigations by cryo-EM. In Aim 2, we will leverage our abilities in isolating HER2 to biophysically characterize the influence of HER2 oncogenic mutations on oligomerization state and structure by EM. We will then correlate the in vitro observations with downstream signaling. The completion of this multidisciplinary project will represent a significant scientific contribution, not only due to the technological advances required to study single-pass transmembrane receptors but also in the light of learning how HER2 regulates itself without ligand or co-receptor. Such knowledge could be applied to developing new drugs, selectively targeting mutant forms of HER2, and counteracting drug resistance common with anti-HER2 therapies.